Open access

Serogroups and genotypes of Leptospira spp. strains from bovine aborted fetuses
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Delooz Laurent1,2, Czaplicki Guy1, Gregoire Fabien1, Dal Pozzo Fabiana2, Pez Floriane3, Kodjo
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Angeli4, Saegerman Claude2
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1 Regional Association for Animal Registration and Health (ARSIA) asbl, 5590 Ciney, Belgium
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2 Research Unit of Epidemiology and Risk Analysis applied to veterinary science (UREAR-
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ULg), Fundamental and Applied Research for Animals & Health (FARAH) Center, Faculty of
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Veterinary Medicine, University of Liege, 4000 Liege, Belgium
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3 BioSellal, 69007 Lyon, France
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4 Laboratoire des Leptospires, VetAgro Sup, Campus Vétérinaire de Lyon, 69280 Marcy-
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l’Etoile, France
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ABSTRACT
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Leptospirosis is a global disease of animals, with potential major economic impact on livestock
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industry and important zoonotic capacities. The disease represents a major challenge in the
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developing countries as humans and animals frequently live in close association. The serovar
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Hardjo of Leptospira whose primary host is cattle has been studied extensively, but few data
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exist on other current circulating or emerging serotypes. To better understand the disease in
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cattle and how to prevent and/or control it, it is necessary to identify the genotype and the
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serotype of circulating Leptospira. This paper presents results of several investigations
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performed on a historical Belgian collection of congenital jaundice in bovine aborted fetuses
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coming from the leptospirosis emerging episode of 2014 (Delooz et al., 2015). The results
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revealed that L. Grippotyphosa and L. Australis were the most prevalent serogroups with
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respectively 17/42 and 13/42 positive MAT during this emerging event associated with the
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same clinical pattern. The study also confirms that congenital jaundice is associated with L.
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kirscheneri and L. interrogans and provides the genotyping of DNA obtained from these two
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species.
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Keywords: Abortion, Cattle, Icteric, Jaundice, Genotyping, Leptospira, Leptospirosis,
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Belgium
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INTRODUCTION
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In Belgium, a national surveillance program based on the compulsory reporting of
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abortions and subsequent analyses on their products reached several objectives including
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official surveillance of bovine brucellosis but also the monitoring of other bovine abortive
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diseases. Some emerging or re-emerging pathogens could be identified, including Bluetongue
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virus serotype 8, Brucella abortus and more recently Schmallenberg virus.
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Since July 2014, the Belgian Walloon region faced an unexpected situation with a
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drastic increase of congenital jaundice in bovine aborted fetuses (Delooz et al., 2015). During
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the last six years, abortions associated with jaundice had been notified but the monthly
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incidence of cases remained stable. From July to December 2014, an increase of bovine aborted
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fetuses with jaundice was reported by ARSIA pathologists, with a significantly higher incidence
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than previous months and years. The standardized panel of analyses designed to extend the
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diagnosis did not allow identifying the etiology. After additional analyzes, high levels of
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antibodies against Leptospira serogroups Australis and Grippotyphosa were found in cows after
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abortion of icteric fetuses. Serology performed during the emergence identified serogroups
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without providing information on the genotype of the involved pathogenic Leptospira strains.
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A leptospiral infection was consequently hypothezised (Delooz et al., 2015).
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Leptospirosis is a transmissible disease of animals and humans caused by the spirochete
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Leptospira. All the pathogenic leptospires were formerly classified as members of the species
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Leptospira interrogans. However, the genus has been reorganised and pathogenic Leptospira
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are now classified in 23 species (Adler, 2010; Levett, 2001; Morey et al., 2006) from which
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more than 300 distinct serovars included within 24 serogroups are distinguished (Levett, 2015).
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Laboratory diagnosis of leptospirosis can be complex and involves tests designed to detect
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specific antibodies against Leptospira, as well as for direct isolation of leptospires, antigens
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detection and detection of Leptospira nucleic acid in animal tissues or body fluids (OIE, 2014).
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In cattle, the seroprevalence of the serogroup Sejroe is one of the most studied and varies
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widely from one country to another with 33% in France (Ayral et al., 2014), 30% in the
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Netherlands (Hartman et al., 1989) and up to 83.59% in Ireland (Ryan et al., 2012). In addition,
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antibodies against Leptospira serovar Hardjo were found in tank milk of 9.2% bovine dairy
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herds, with a higher incidence in the southern part of the country (Dom et al., 1991). In France,
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microscopic agglutination test (MAT) performed on samples collected in 394 cattle herds
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allowed to determine the distribution of the following three predominant circulating serogroups
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(Australis, Sejroe, and Grippotyphosa) (Ayral et al., 2014). In this part of Europe, leptospirosis
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is prevalent and may be responsible for pathological events in human (Mori et al., 2014) and
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animal health (Delooz et al., 2015).
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Leptospirosis is a known cause of abortions and infection can be accompanied by a wide
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variety of clinical signs. However, fetal jaundice was not observed during experimental
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infections (Ellis and Michma, 1977; Ellis et al., 1986; Smith et al., 1997). On the contrary based
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on the recent field observations, the hypothesis of the association of bovine fetal jaundice with
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leptospirosis was formulated (Delooz et al., 2015). Currently, little epidemiological information
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exists concerning the different serovars of Leptospira circulating among cattle in Belgium.
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While the manifestations of the disease can be very different depending on the strain and the
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animal species, it is important to identify the pathogenic strain in order to tackle the best
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prevention and control measures and thereby, prevent potential transmission to humans
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(Evangelista and Coburn, 2010).
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The aim of this work was to characterize the Leptospira infection detected in bovine
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abortion cases associated with fetal jaundice which occurred in Southern Belgium in 2014.
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MATERIAL AND METHODS
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Study desing
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In the context of the Belgian passive surveillance program for bovine brucellosis, a total
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of 42 bovine abortion cases collected from October 2011 to December 2014 were included in
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this study. They originated from 39 cattle farms distributed among the 5 Walloon provinces.
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They were included in the study according to the diagnosis of congenital jaundice (N = 41) or
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the PCR positivity against pathogenic Leptospira (N = 1).
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Information issued from the anamnesis, such as sampling date, herd identification
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number, cattle breed, month of pregnancy and number of parity were encoded in the laboratory
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information management system (LIMS) or in an Access database. None of these herds
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applied vaccination against Leptospira species and, moreover, no Leptospira vaccine has a
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marketing authorization in Belgium. The geographical localization of each case of abortion was
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possible using the Lambert coordinates and the Belgian cattle identification and movement
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traceability system (SANITRACE).
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Laboratory analyses
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A standardized panel of analyses was first applied to perform the laboratory diagnosis
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of bovine abortion on submitted fetuses. Direct and/or indirect detection of pathogens was
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performed, including bacteria (Brucella spp., Campylobacter spp., Coxiella burnetii,
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Leptospira borgpetersenii and interrogans serovar Hardjo, Listeria monocytogenes, Neospora
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caninum, Salmonella Dublin), viruses (bluetongue virus serotype 8 (BTV-8), bovine
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herpesvirus 1 (BoHV-1), bovine herpesvirus 4 (BoHV-4), bovine viral diarrhea virus (BVDV),
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Schmallenberg virus), several mycotic agents, and many other opportunistic bacteria (Table I).
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Microscopic agglutination tests
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MAT was performed on the 42 maternal sera sampled on the aborted cows at the time of
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abortion using twenty-four serovars representing 14 serogroups of pathogenic Leptospira
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species: Icterohaemorrhagiae, Copenhageni, Australis, Bratislava, Munchen, Autumnalis, Bim,
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Castellonis, Bataviae, Canicola, Hebdomadis, Panama, Mangus, Pomona, Mozdok, Pyrogenes,
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Sejroe, Saxkoebing, Hardjo, Wolffi, Tarassovi, Cynopteri, Vanderhoedoni, and Grippotyphosa
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(Table II). According to observations recorded by Chappel and collaborators in 2004, titers of
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160 or higher were defined as positive agglutination reactions for ruminants. The end-point was
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the highest dilution of serum in which 50% agglutination still occured.
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Pathogenic Leptospira DNA detection (real-time PCR).
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During the necropsy, a spleen, kidney, liver and placenta fragment were sampled on
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each abortion cases, pooled and stored at -20°C. PCR analysis was performed on 26 pools of
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organs sampled from icteric fetuses, retrieved in the historical abortion samples collection
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described in the study design (not available for 16 other fetuses). DNA extraction was
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performed using KingFisher TM Flex 96 Magnetic Particle Processors (Thermo Scientific TM,
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UK) and LSI MagVet TM Universal Isolation Kit (Life Technologies, UK) and was followed by
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pathogenic Leptospira DNA detection using a commercial PCR test (TaqVet TM PathoLept TM,
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Thermofisher, France) on organ pool according to the manufacturer's instructions (Levett et al.,
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2005).
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